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Nucleic Acids Research
Article . 2001 . Peer-reviewed
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Detection of the 5'-cap structure of messenger RNAs with the use of the cap-jumping approach

Authors: V A, Efimov; O G, Chakhmakhcheva; J, Archdeacon; J M, Fernandez; O N, Fedorkin; Y L, Dorokhov; J G, Atabekov;

Detection of the 5'-cap structure of messenger RNAs with the use of the cap-jumping approach

Abstract

An effective procedure for specific determination of the cap structure at the 5'-terminus of mRNA and for isolation of the corresponding full-length cDNA has been developed. The procedure involves covalent attachment of an oligonucleotide template extender to the 5'-cap structure of mRNA followed by RT-PCR using M-MLV SuperScript II reverse transcriptase. In the course of reverse transcription, the enzyme 'jumps over' the cap structure and includes the sequence complementary to the oligonucleotide template extender into the 3'-end of the first cDNA strand. The cap-jumping method was successfully tested using some mammalian cellular mRNAs, genomic RNAs of tobacco mosaic virus (TMV) U1 and the recently isolated crucifer-infecting tobamovirus. Moreover, cDNA products corresponding to the genomic tobamovirus RNA were obtained from total RNA extracted from tobacco plants infected by crucifer-infecting tobamovirus or tobacco mosaic virus. Using the cap-jumping method, we have shown for the first time that genomic crucifer-infecting tobamovirus (crTMV) RNA contains a 5'-cap structure. This improved method can be recommended for the construction of full-length and 5'-end enriched cDNA libraries, identification of capped RNAs and determination of their 5'-terminal sequences.

Keywords

RNA Caps, DNA, Complementary, Base Sequence, Reverse Transcriptase Polymerase Chain Reaction, Tobamovirus, Oligonucleotides, RNA-Directed DNA Polymerase, Globins, Animals, RNA, Viral, RNA, Messenger, Rabbits, 5' Untranslated Regions

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selected citations
These citations are derived from selected sources.
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
15
Average
Top 10%
Average
gold